Methods and apparatus for creating particle derivatives of HDL with reduced lipid content

ABSTRACT

The present invention is directed to systems, apparatus and methods for creating derivatives of at least one form of HDL without substantially affecting LDL. These derivatives of HDL are particles with reduced lipid content, particularly reduced cholesterol content. These particles have the capacity to bind cholesterol and are administered to a patient to enhance cellular cholesterol efflux and reduce cholesterol levels in cells, tissues, organs, and blood vessels. The present method is useful for treating atherogenic vascular disease and may be combined with other therapies such as statins, inhibitors of cholesterol absorption, niacin, anti-inflammatories, exercise and dietary restriction.

PRIOR RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.provisional patent application Ser. No. 60/484,690 filed Jul. 3, 2003.

FIELD OF INVENTION

The present invention generally relates to systems, apparatus andmethods for removing lipids from HDL particles while leaving LDLparticles substantially intact, via the extracorporeal treatment ofblood plasma using either a single solvent or multiple solvents. Themethod of the present invention provides a procedure for selectiveremoval of lipid from HDL to create a modified HDL particle whileleaving LDL particles substantially intact. The method of the presentinvention provides a procedure for removing LDL particles from plasmabefore treating the HDL particles to remove lipid. This inventioncreates particles comprising derivatives of HDL that may be administeredto an animal or human for therapeutic use such as treatment ofarteriosclerosis and atherosclerotic vascular diseases within an animalor human.

BACKGROUND

Introduction—Hyperlipidemia and Arteriosclerosis

Cardiovascular, cerebrovascular, and peripheral vascular diseases areresponsible for a significant number of deaths annually in manyindustrialized countries. One of the most common pathological processesunderlying these diseases is arteriosclerosis. Arteriosclerosis ischaracterized by lesions, which begin as localized fatty thickenings inthe inner aspects of blood vessels supplying blood to the heart, brain,and other organs and tissues throughout the body. Over time, theseatherosclerotic lesions may ulcerate, exposing fatty plaque depositsthat may break away and embolize within the circulation. Atheroscleroticlesions obstruct the lumens of the affected blood vessels and oftenreduce the blood flow within the blood vessels, which may result inischemia of the tissue supplied by the blood vessel. Embolization ofatherosclerotic plaques may produce acute obstruction and ischemia indistal blood vessels. Such ischemia, whether prolonged or acute, mayresult in a heart attack or stroke from which the patient may or may notrecover. Similar ischemia in an artery supplying an extremity may resultin gangrene requiring amputation of the extremity.

For some time, the medical community has recognized the relationshipbetween arteriosclerosis and levels of dietary lipid, serum cholesterol,and serum triglycerides within a patient's blood stream. Manyepidemiological studies have been conducted revealing that the amount ofserum cholesterol within a patient's blood stream is a significantpredictor of coronary disease. Similarly, the medical community hasrecognized the relationship between hyperlipidemia and insulinresistance, which can lead to diabetes mellitus. Further, hyperlipidemiaand arteriosclerosis have been identified as being related to othermajor health problems, such as obesity and hypertension.

Cholesterol Transport

Cholesterol circulating in the blood is carried by plasma lipoproteinsthat transport lipids throughout the blood. The plasma lipoproteins areclassified in five types according to size: chylomicrons (which arelargest in size and lowest in density), very low-density lipoproteins(VLDL), intermediate density lipoproteins (IDL), low-densitylipoproteins (LDL), and high-density lipoproteins (HDL) (which are thesmallest and most dense). These plasma lipoproteins exhibit differencesin size, density, diameter, protein content, phospholipid content andtriacylglycerol content, known to one of ordinary skill in the art. Ofthese, the low-density lipoprotein (LDL) and high-density lipoprotein(HDL) are primarily the major cholesterol carrier proteins. The proteincomponent of LDL, the apolipoprotein-B (Apo B) and its products comprisethe atherogenic elements. Elevated plasma LDL levels and reduced HDLlevels are recognized as the primary cause of coronary disease becauseApo B is in highest concentration in LDL particles and is not present inHDL particles. Apolipoprotein A-1 (Apo A-1) and apolipoprotein A-2 (ApoA-2) are found in HDL. Other apolipoproteins, such as Apo C and itssubtypes (C-1, C-2 and C-3), Apo D and Apo E are also found in HDL. ApoC and Apo E are also observed in LDL particles.

Numerous major classes of HDL particles including HDL_(2b), HDL_(2a),HDL_(3a), HDL_(3b) and HDL_(3c) have been reported (Segrest et al.,Curr. Opin. Lipidol. 11:105-115, 2000). Various forms of HDL particleshave been described on the basis of electrophoretic mobility on agaroseas two major populations, a major fraction with α-HDL mobility and aminor fraction with migration similar to VLDL (Barrans et al.,Biochemica Biophysica Acta 1300; 73-85, 1996). This latter fraction hasbeen called pre-β HDL and these particles are thought to be the mostefficient HDL particle subclass for inducing cellular cholesterol efflux(Segrest et al., Curr. Opin. Lipidol. 11:105-115, 2000). The pre-β HDLparticles have been further separated into pre-β₁ HDL, pre-β₂ HDL andpre-β₃ HDL. These lipoprotein particles are comprised of Apo A-1,phospholipids and free cholesterol. The pre-β HDL particles areconsidered to be the first acceptors of cellular free cholesterol andare essential in eventually transferring free and esterified cholesterolto α-HDL (Barrans et al., Biochemica Biophysica Acta 1300; 73-85, 1996).Pre-β₃ HDL particles may transfer cholesterol to α-HDL or be convertedto α-HDL. These pre-β HDL particles have been characterized in terms oftheir charge, molecular mass (ranging from 40 kDa-420 kDa), size(Stoke's radius 4 nm-15 nm), shape (ellipsoidal, discoidal or spherical)and chemical composition (protein (including Apo A-1), free cholesterol,esterified cholesterol, phospholipids and the ratio of free cholesterolto phospholipids (see Barrans et al., Biochemica Biophysica Acta 1300;73-85, 1996 for additional details)). HDL levels are inverselycorrelated with atherosclerosis and coronary artery disease.Accordingly, what is needed is a method to decrease or removecholesterol from these various HDL particles, especially the pre-β HDLparticles, so that they are available to remove additional cholesterolfrom cells.

Cholesterol is synthesized by the liver or obtained from dietarysources. LDL is responsible for transferring cholesterol from the liverto tissues at different sites in the body. However, if LDL collects onthe arterial walls, it undergoes oxidation caused by oxygen freeradicals liberated from the body's chemical processes and interactsdeleteriously with the blood vessels. The modified LDL causes whiteblood cells in the immune system to gather at the arterial walls,forming a fatty substance called plaque and injuring cellular layersthat line blood vessels. The modified oxidized LDL also reduces thelevel of nitric oxide, which is responsible for relaxing the bloodvessels and thereby allowing the blood to flow freely. As this processcontinues, the arterial walls slowly constrict, resulting in hardeningof the arteries and thereby reducing blood flow. The gradual build-up ofplaque can result in blockage of a coronary vessel and ultimately in aheart attack.

In contrast to LDL, high plasma HDL levels are desirable because theyplay a major role in “reverse cholesterol transport”, where the excesscholesterol is transferred from tissue sites to the liver where it iscatabolized and eliminated. Optimal total cholesterol levels are 200mg/dl or below with a LDL cholesterol level of 160 mg/dl or below and aHDL-cholesterol level of 45 mg/dl for men and 50 mg/dl for women. LowerLDL levels are recommended for individuals with a history of elevatedcholesterol, atherosclerosis or coronary artery disease.

Current Methods of Treatment

Hyperlipidemia may be treated by changing a patient's diet. However,diet as a primary mode of therapy requires a major effort on the part ofpatients, physicians, nutritionists, dietitians, and other health careprofessionals and thus undesirably taxes the resources of healthprofessionals. Another negative aspect of this therapy is that itssuccess does not rest exclusively on diet. Rather, success of dietarytherapy depends upon a combination of social, psychological, economic,and behavioral factors. Thus, therapy based only on correcting flawswithin a patient's diet is not always successful.

In instances when dietary modification has been unsuccessful, drugtherapy has been used as an alternative. Such therapy has included useof commercially available hypolipidemic drugs administered alone or incombination with other therapies as a supplement to dietary control.These drugs, called statins, include natural statins, lovastatin,pravastatin, simvastatin, fluvastatin, atorvastatin, and cerivastatin.Statins are particularly effective for lowering LDL levels and are alsoeffective in the reduction of triglycerides, apparently in directproportion to their LDL-lowering effects. Statins raise HDL levels, butto a lesser extent than other anti-cholesterol drugs. Statins alsoincrease nitric oxide, which, as described above, is reduced in thepresence of oxidized LDL.

Bile acid resins, another drug therapy, work by binding with bile acid,a substance made by the liver using cholesterol as one of the primarymanufacturing components. Because the drugs bind with bile acids in thedigestive tract, they are then excreted with the feces rather than beingabsorbed into the body. The liver, as a result, must take morecholesterol from the circulation to continue constructing bile acids,resulting in an overall decrease in LDL levels.

Nicotinic acid, or niacin, is also known as vitamin B₃. It is extremelyeffective in reducing triglyceride levels and raising HDL levels higherthan any other anti-cholesterol drug. Nicotinic acid also lowersLDL-cholesterol.

Fibric acid derivatives, or fibrates, are used to lower triglyceridelevels and increase HDL when other drugs ordinarily used for thesepurposes, such as niacin, are not effective.

Probucol lowers LDL-cholesterol levels, however, it also lowers HDLlevels. It is generally used for certain genetic disorders that causehigh cholesterol levels, or in cases where other cholesterol-loweringdrugs are ineffective or cannot be used.

Hypolipidemic drugs have had varying degrees of success in reducingblood lipid; however, none of the hypolipidemic drugs successfullytreats all types of hyperlipidemia. While some hypolipidemic drugs havebeen fairly successful, the medical community has not found anyconclusive evidence that hypolipidemic drugs cause regression ofatherosclerosis. In addition, all hypolipidemic drugs have undesirableside effects. As a result of the lack of success of dietary control,drug therapy and other therapies, atherosclerosis remains a major causeof death in many parts of the world.

New therapies have been used to reduce the amount of lipid in patientsfor whom drug and diet therapies were not sufficiently effective. Forexample, extracorporeal procedures like plasmapheresis and LDL-apheresishave been employed and are shown to be effective in lowering LDL.

Plasmapheresis therapy or plasma exchange therapy, involves replacing apatient's plasma with donor plasma or more usually a plasma proteinfraction. Plasmapheresis is a process whereby the blood plasma isremoved from blood cells by a cell separator. The separator works eitherby spinning the blood at high speed to separate the cells from the fluidor by passing the blood through a membrane with pores so small that onlythe fluid component of the blood can pass through. The cells arereturned to the person undergoing treatment, while the plasma isdiscarded and replaced with other fluids.

This treatment has resulted in complications due to the introduction offoreign proteins and transmission of infectious diseases. Further,plasmapheresis has the disadvantage of non-selective removal of allserum proteins, such as VLDL, LDL, and HDL. Moreover, plasmapheresis canresult in several side effects including allergic reactions in the formof fever, chills, and rash and possibly even anaphylaxis.

As described above, it is not desirable to remove HDL, which is secretedfrom both the liver and the intestine as nascent, disk-shaped particlesthat contain cholesterol and phospholipids. HDL is believed to play arole in reverse cholesterol transport, which is the process by whichexcess cholesterol is removed from tissues and transported to the liverfor reuse or disposal in the bile.

In contrast to plasmapheresis, the LDL-apheresis procedure selectivelyremoves Apo B containing cholesterol, such as LDL, while retaining HDL.Several methods for LDL-apheresis have been developed. These techniquesinclude absorption of LDL in heparin-agarose beads, the use ofimmobilized LDL-antibodies, cascade filtration absorption to immobilizedextran sulphate, and LDL precipitation at low pH in the presence ofheparin. Each method described above is effective in removing LDL. Thistreatment process has disadvantages, however, including the failure topositively affect HDL or to cause a metabolic shift that can enhanceatherosclerosis and other cardiovascular diseases. LDL apheresis merelytreats patients with severe hyperlipidemia.

Yet another method of achieving a reduction in plasma cholesterol inhomozygous familial hypercholesterolemia, heterozygous familialhypercholesterolemia and patients with acquired hyperlipidemia is anextracorporeal lipid elimination process, referred to as cholesterolapheresis. In cholesterol apheresis, blood is withdrawn from a patient,the plasma is separated from the blood, and the plasma is mixed with asolvent mixture. The solvent mixture extracts lipids from the plasma.Thereafter, the delipidated plasma is recombined with the patient'sblood cells and returned to the patient.

Conventional extracorporeal delipidation processes, however, aredirected toward the concurrent delipidation of LDL and HDL. This processcan have a number of disadvantages. Because LDL is more difficult todelipidate, extracorporeal systems are designed to subject body fluidvolumes to substantial processing, possibly through multiple stagesolvent exposure and extraction steps. Vigorous multi-stage solventexposure and extraction can have several drawbacks. It may be difficultto remove a sufficient amount of solvents from the delipidated plasma inorder for the delipidated plasma to be safely returned to a patient.

Hence, existing apheresis and extracorporeal systems for treatment ofplasma constituents suffer from a number of disadvantages that limittheir ability to be used in clinical applications. A need exists forimproved systems, apparatuses and methods capable of removing lipidsfrom blood components in order to provide treatments and preventativemeasures for cardiovascular diseases. What is also needed is a method toselectively remove lipid from HDL particles and thereby create modifiedHDL particles with increased capacity to accept cholesterol. What isalso needed is a method to selectively remove lipid from HDL particlesand thereby create modified HDL particles with increased capacity toaccept cholesterol, without substantially affecting LDL particles.

SUMMARY OF THE INVENTION

The present invention is directed to systems, apparatus and methods forcreating modified HDL particles without substantially affecting LDL.These modified HDL particles are derivatives of HDL with reduced lipidcontent, particularly reduced cholesterol content. These modified HDLparticles have the capacity to bind cholesterol and may be administeredto a patient to enhance cellular cholesterol efflux and reducecholesterol levels in cells, tissues, organs and blood vessels.

The present invention also provides a biological fluid comprising amodified protein distribution wherein the biological fluid had a firststate, the first state having alpha high density lipoproteins andpre-beta high density lipoproteins, and wherein the biological fluid hasa second state, the second state having an increased concentration ofpre-beta high density lipoprotein relative to the first state, afterbeing exposed to a lipid removing agent.

The present invention also provides a biological fluid capable ofenhancing an ABCA1 pathway of a patient wherein the biological fluid ismade by modifying a fluid having a first concentration of pre-beta highdensity lipoproteins relative to total protein, wherein the modificationincreases the concentration of pre-beta high density lipoproteinrelative to total protein.

The present invention further provides a method of enhancing an ABCA1pathway of a patient with a first protein distribution, the firstprotein distribution having a concentration of pre-beta high densitylipoproteins relative to total protein, comprising the step of modifyinga fluid containing the first protein distribution by exposing the fluidto a lipid removing agent, wherein the modification increases theconcentration of pre-beta high density lipoprotein relative to totalprotein, and introducing the fluid into the patient.

The present invention further provides a method of modifying a proteindistribution in a fluid wherein the protein distribution has a firststate, said first state having alpha high density lipoproteins andpre-beta high density lipoproteins, comprising the steps of: exposingsaid fluid to a lipid removing agent wherein the exposure modifies theprotein distribution from the first state into a second state, saidsecond state having an increased concentration of pre-beta high densitylipoprotein relative to said first state; and removing said lipidremoving agent from the biological fluid.

The present invention discloses a method for removing lipids fromfluids, such as blood plasma, and from HDL particles withoutsubstantially affecting LDL by treating the fluid with solvents andadding energy to mix the solvents and fluid. Removing lipid from HDLparticles creates a modified HDL particle with reduced lipid content,which is capable of binding additional lipid and enhancing cellularcholesterol efflux. More particularly, the present invention is directedtoward removal of lipids from HDL particles in blood plasma using eithera single solvent or multiple solvents, thereby creating new particlesthat are derivatives of HDL with reduced lipid content.

In one embodiment of the present invention, LDL and HDL particles areseparated prior to treatment of the plasma containing the HDL particles.LDL is extracted and the plasma is treated to reduce the lipid contentof HDL particles. Subsequent to LDL removal, the plasma containing HDLparticles is exposed to lipid removing agents using the present methodsto reduce lipid levels and create particle derivatives of HDL withreduced lipid content. These particles demonstrate enhanced capacity forbinding cholesterol. These particle derivatives of HDL and the plasmawith reduced lipid content may be administered to the patient in orderto enhance cellular cholesterol efflux and treat lipid-associateddiseases and conditions.

In another embodiment of the present invention, the LDL is retained (notseparated prior to treatment) and a solvent system is employed toselectively remove lipid from HDL and create particles comprised ofderivatives of HDL with reduced lipid content while not substantiallyaffecting LDL. The separated plasma is mixed with a solvent systemdesigned to selectively decrease lipid in HDL particles present in theplasma. Care is taken to ensure that the solvent employed, the mixingmethod employed, procedure, mixing time, and temperature create anoptimal solvent system that will selectively remove lipid from HDL,create particles comprised of derivatives of HDL, and leave LDLsubstantially intact. The at least partially or substantiallydelipidated plasma, which was separated initially, is then treatedappropriately for administration to a patient.

The present invention may be employed to treat plasma obtained from apatient for subsequent administration to the patient or foradministration into another patient. The present invention may also beused to treat blood and plasma stored in blood banks in order to createplasma with reduced lipid content and containing particles comprised ofderivatives of HDL with reduced lipid content. This treated plasmacontaining particles comprised of derivatives of HDL with reduced lipidcontent may be used for heterologous administration to anotherindividual in order to enhance cholesterol efflux in the patient. Thepresent invention may also be employed to create particles comprised ofderivatives of HDL that may be collected and stored.

The present method modifies various forms of different HDL particles.Such HDL particles include but are not limited to those HDL particlesthat have been described based on a variety of methods such as methodsthat measure charge, density, size and immunoaffinity, including but notlimited to electrophoretic mobility, ultracentrifugation,immunoreactivity and other methods known to one of ordinary skill in theart. Such HDL particles include but are not limited to the following:VLDL, α HDL, pre-β HDL (including pre-β₁ HDL, pre-β₂ HDL and pre-β₃HDL), β HDL, HDL₂ (including HDL_(2a) and HDL_(2b)), HDL₃, VHDL, LpA-I,LpA-II, LpA-I/LpA-II (for a review see Barrans et al., BiochemicaBiophysica Acta 1300; 73-85, 1996). Accordingly, practice of the methodsof the present invention creates modified HDL particles. These modifiedHDL particles may be modified in numerous ways, including but notlimited to changes in one or more of the following metabolic and orphysicochemical properties: molecular mass (kDa); charge; diameter;shape; density; hydration density; flotation characteristics; content ofcholesterol; content of free cholesterol; content of esterifiedcholesterol; molar ratio of free cholesterol to phospholipids;immunoaffinity; content, activity or helicity of one or more of thefollowing enzymes or proteins (Apo A-1, Apo A-2, Apo D, Apo E, Apo J,Apo A-IV, cholesterol ester transfer protein (CETP),lecithin:cholesterol acyltransferase (LCAT); capacity and/or rate forcholesterol binding, capacity and/or rate for cholesterol transport. Thephysical-chemical properties of HDL particles are known to one ofordinary skill in the art. For example, pre-β HDL particles have beencharacterized in terms of their charge, molecular mass (ranging from 40kDa-420 kDa), size (Stoke's radius 4 nm-15 nm), shape (ellipsoidal,discoidal or spherical) and chemical composition (protein (including ApoA-1), free cholesterol, esterified cholesterol, phospholipids and theratio of free cholesterol to phospholipids (see Barrans et al.,Biochemica Biophysica Acta 1300; 73-85, 1996 for additional details)).

The present invention creates these modified HDL particles withoutsubstantially affecting various metabolic and or physico-chemicalproperties of LDL particles.

In another aspect of the present invention, the modified HDL derivativeparticles made with the disclosed method are administered to a patientin order to enhance cholesterol efflux from cells. These modified HDLparticles may be obtained from the same patient or a different patientwho will receive the modified HDL particles. These particles may becombined with plasma treated with the methods of the present inventionand containing substantially reduced levels of lipid and thenadministered to a patient.

The present invention also provides a modified Apo A-1 protein producedby treating plasma with the method of the present invention, wherein themodified Apo A-1 protein has reduced lipid content. The modified Apo A-1protein is purified and may be administered to a patient either alone orin conjunction with the modified HDL particles with reduced lipidcontent to enhance cholesterol efflux.

These modified HDL particles may also be combined with heterologousplasma treated with the methods of the present invention and containingsubstantially reduced levels of lipid and then administered to apatient. These particles may be combined with other plasma constituents,and optionally with red blood cells before administration into thevascular system. Administration of these particles occurs as frequentlyas necessary to effectuate cholesterol efflux from cells.

The modified HDL particles of the present invention are administered topatients in order to reduce cellular levels of cholesterol, and areindicated for a variety of conditions, including but not limited toatherosclerosis, arteriosclerosis, hyperlipidemia, hypercholesterolemia,obesity, hypertension, stroke, neuroprotection following stroke,inflammation, Alzheimer's disease, diabetes, low endogenous HDL levels,high LDL levels, cardiovascular disease (including atherosclerosis ofthe coronary arteries, carotid arteries, subclavian, brachial, aorta,iliac, renal, femoral, popliteal, tibial or any other artery in thecardiovascular system), cerebrovascular disease (includingatherosclerosis of the internal carotid, middle cerebral, anteriorcerebral, posterior cerebral, basilar, cerebellar, and/or spinalarteries, or any branch of these arteries, cerebral cortical endarteries, or any other artery supplying the central nervous system).

The modified HDL particles of the present invention are administered toa patient according to any schedule that is effective to enhancecellular cholesterol efflux. In one non-limiting example, a liter ofplasma is treated with the methods of the present invention each weekand the treated plasma containing the modified HDL particles is returnedto the patient each week for four to six weeks. Alternatively, themodified HDL particles may be separated from the treated plasma andadministered in an acceptable vehicle.

It is to be understood that the modified HDL particles of the presentinvention may be administered in conjunction other regimens andtreatments for treatment of the diseases and conditions mentioned above.For example, the modified HDL particles of the present invention may beadministered in conjunction with exercise and/or dietary restriction offat and cholesterol intake.

The modified HDL particles of the present invention may be used inconjunction with administration of agents for reducing cholesterol,reducing LDL levels and enhancing HDL levels. These agents, such asHMG-CoA reductase inhibitors, or statins, may be administered in dosagesand according to administration schedules commonly known to one ofordinary skill in the art. Statins include but are not limited tocerivastatin, atorvastatin, fluvastatin, simvastatin, pravastatin andlovastatin. For example, dosages of 10 mg, 20 mg, 40 mg or 80 mg ofstatins, taken once per day, are commonly employed. Administration ofthe modified HDL particles of the present invention can eliminate theneed for statin therapy in patients or reduce the required dosage ofstatins.

In another aspect, the modified HDL particles of the present inventionare used in conjunction with administration of agents designed to reduceabsorption of fat and cholesterol. Such agents, for example ezetimibe,and the clinically appropriate dosages are known to one of ordinaryskill in the art.

In yet another aspect of the present invention, the modified HDLparticles are used in conjunction with administration of one or moreagents such as fibric acid derivatives (gemfibrozil), nicotinic acid(niacin), and bile acid-binding resins (cholestyramine, cholestipol).

In yet another aspect, the modified HDL particles of the presentinvention are used in conjunction with administration ofanti-inflammatory drugs known to one of ordinary skill in the art, suchas aspirin. Anti-inflammatory drugs are often prescribed to patientswith vascular disease since it is believed that inflammation is acausative factor of atherosclerosis and other vascular diseases.

The modified HDL particles of the present invention are used inconjunction with administration of agents such as statins together withagents designed to reduce absorption of fat and cholesterol. Thiscombination of three therapies is effective in enhancing cholesterolefflux from cells and permits administration of lower dosages ofstatins. The modified HDL particles of the present invention are alsoused in conjunction with any of the therapeutic approaches describedabove.

These modified HDL particles may be stored before use. They may be madefrom a patient's plasma and returned to that patient. Alternatively, themodified HDL particles may be made from plasma obtained from a firstpatient and subsequently administered to a second patient. The presentinvention is useful in creating plasma samples containing modified HDLparticles for storage in a plasma bank and subsequent administration topatients.

Accordingly, it is an object of the present invention to provideparticles comprising modified HDL particles.

It is another object of the present invention to provide particlescomprising modified HDL particles without substantially affecting LDL.

Yet another object of the present invention is to provide particlescomprising derivatives of at least one form of HDL, wherein the particlehas a reduced cholesterol content.

It is another object of the present invention to provide particlescomprising derivatives of at least one form of HDL with a reduced ratioof free cholesterol to phospholipid.

Another object of the present invention is to provide particlescomprising derivatives of at least one form of HDL, wherein theparticles are pre-β HDL particles.

Yet another object of the present invention is to provide a biologicalfluid comprising a modified protein distribution wherein the biologicalfluid had a first state, the first state having alpha high densitylipoproteins and pre-beta high density lipoproteins, and wherein thebiological fluid has a second state, the second state having anincreased concentration of pre-beta high density lipoprotein relative tothe first state, after being exposed to a lipid removing agent.

Accordingly, it is an object of the present invention to provide a novelmethod for creation of particles comprising derivatives of at least oneform of HDL.

It is yet another object of the present invention to provide a novelmethod for creation of particles comprising derivatives of at least oneform of HDL without substantially affecting LDL.

Another object of the present invention is to provide a method ofmodifying a protein distribution in a fluid wherein the proteindistribution has a first state, said first state having alpha highdensity lipoproteins and pre-beta high density lipoproteins, comprisingthe steps of: exposing said fluid to a lipid removing agent wherein theexposure modifies the protein distribution from the first state into asecond state, said second state having an increased concentration ofpre-beta high density lipoprotein relative to said first state; andremoving said lipid removing agent from the biological fluid.

It is yet another object of the present invention to provide abiological fluid capable of enhancing an ABCA1 pathway of a patientwherein the biological fluid is made by modifying a fluid having a firstconcentration of pre-beta high density lipoproteins relative to totalprotein, wherein the modification increases the concentration ofpre-beta high density lipoprotein relative to total protein.

Another object of the present invention is to provide a method ofenhancing an ABCA1 pathway of a patient with a first proteindistribution, the first protein distribution having a concentration ofpre-beta high density lipoproteins relative to total protein, comprisingthe step of modifying a fluid containing the first protein distributionby exposing the fluid to a lipid removing agent, wherein themodification increases the concentration of pre-beta high densitylipoprotein relative to total protein, and introducing the fluid intothe patient.

Yet another object of the present invention is to provide a novel methodfor treating diseases associate with lipid accumulation by administeringto a patient a composition comprising particles that are derivatives ofat least one form of HDL.

It is another object of the present invention to provide a novel methodfor treating diseases associated with lipid accumulation byadministering to a patient a composition comprising particles that arederivatives of at least one form of HDL in conjunction with therapeuticadministration of a statin, an inhibitor of cholesterol or lipid uptake,niacin, fibric acid derivatives, bile acid-binding resins, or acombination thereof.

Yet another object of the present invention is to provide a novel methodfor enhancing cellular cholesterol efflux in a patient comprisingadministration of a composition comprising particles that arederivatives of at least one form of HDL.

Still another object of the present invention is to provide a novelmethod for treating atherosclerosis by administering to a patient acomposition comprising particles that are derivatives of at least oneform of HDL.

Another object of the present invention is to provide a kit useful fortreating a biological fluid in order to reduce cholesterol and lipid andto create particles comprising derivatives of at least one form of HDL.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart delineating the steps of the LDL extraction, andsubsequent creation of modified HDL particles.

FIG. 2 is a flowchart delineating the steps of the selective creation ofmodified HDL particles.

FIG. 3 is a schematic of an FPLC profile of a sample of plasma from apool of normal plasma. Total cholesterol (TC) is represented as acontinuous line; phospholipid (PPL) is the dashed line; apolipoproteinA1 (Apo A-1) is the dashed line with a symbol; and, apolipoprotein B(Apo B) is represented by the line with a triangle. Shown are theamounts of these compounds (mg/dl) in each FPLC fraction.

FIG. 4 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent of 100% diisopropyl ether(DIPE) to remove lipid from HDL. Total cholesterol (TC) in the normalsample from FIG. 3 is represented here as a dashed line with triangles.TC in the normal plasma subjected to DIPE is shown as a solid line. ApoA-1 in the normal sample from FIG. 3 is represented here as a solid linewith square symbols. Apo A-1 in the normal plasma subjected to DIPE isshown as a dashed line with a dot symbol.

FIG. 5 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent of 100% DIPE to removelipid from HDL. Apo A-1 in the normal plasma sample from FIG. 3 isrepresented here as a solid line with square symbols. Apo A-1 in thenormal plasma sample subjected to DIPE is shown as a dashed line with adot symbol. Phospholipid (PPL) in the normal plasma sample from FIG. 3is represented here with the dashed line. PPL in the normal plasmasubjected to DIPE is shown as a solid line.

FIG. 6 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent of 100% DIPE to removelipid from HDL. Apo B in the normal plasma sample is represented by theline with a dot symbol. Apo B in the normal plasma subjected to DIPE isshown as dashed line with triangles. Phospholipid (PPL) in the normalsample from FIG. 3 is represented with the solid line. PPL in the normalplasma subjected to DIPE is shown as a dashed line.

FIG. 7 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent ratio of 95:5 sevofluraneto n-butanol. Total cholesterol (TC) in the normal plasma sample fromFIG. 3 is represented here as a dashed line. TC in the normal plasmasubjected to sevoflurane:n-butanol is shown as a solid line. Apo A-1 inthe normal plasma sample from FIG. 3 is represented as a solid line withsquare symbols. Apo A-1 in the normal plasma subjected tosevoflurane:n-butanol is shown as a dashed line with a dot symbol.

FIG. 8 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent ratio of 95:5 sevofluraneto n-butanol. Apo A-1 in the normal sample from FIG. 3 is represented asa solid line with square symbols. Apo A-1 in the normal plasma subjectedto sevoflurane:n-butanol is shown as a dashed line with a dot symbol.Phospholipid (PPL) in the normal sample from FIG. 3 is represented herewith the dashed line. PPL in the normal plasma subjected tosevoflurane:n-butanol is shown as a solid line.

FIG. 9 is a schematic of an FPLC profile of an aliquot from the pool ofnormal plasma which was subjected to a solvent ratio of 95:5 sevofluraneto n-butanol. Apo B in the normal sample from FIG. 3 is represented bythe line with a dot symbol. Apo B in the normal plasma subjected tosevoflurane:n-butanol is shown as dashed line with triangles.Phospholipid (PPL) in the normal sample from FIG. 3 is represented herewith the solid line. PPL in the normal plasma subjected tosevoflurane:n-butanol is shown as a dashed line.

FIG. 10 is a schematic representation of the effect of treatment of aplasma sample with either DIPE or sevoflurane:butanol on alanineaminotransferase (ALT), alkaline phosphatase (AP), bilirubin-T, sodium,potassium, phosphorus, albumin, globulin and the albumin/globulin (A/G)ratio were analyzed in normal untreated plasma and in plasma treatedwith DIPE or with sevoflurane:n-butanol.

FIG. 11 is a schematic representation of a Superose FPLC profile of anormal plasma sample which acts as a control for comparison to thetreatments in FIGS. 12-15. Total cholesterol (TC) in the normal plasmasample is represented here as a solid line. Phospholipid (PPL) isrepresented with the solid line with circles. Apo B is represented bythe line with open squares. Apo A-1 is represented as a solid line withopen triangle symbols. Apo A-2 is shown as a dashed line with a starsymbol.

FIG. 12 is a schematic representation of a Superose FPLC profile of theeffect of treatment of an aliquot of control plasma sample (FIG. 11)with DIPE (100%). Total cholesterol (TC) is represented here as a solidline. Phospholipid (PPL) is represented with the solid line with solidcircles. Apo B is represented by the line with solid squares. Apo A-1 isrepresented as a solid line with closed triangle symbols. Apo A-2 isshown as a dashed line with a star symbol in a solid square.

FIG. 13 is a schematic representation of a Superose FPLC profile of theeffect of treatment of an aliquot of control plasma sample (FIG. 11)with a solvent ratio of 95:5 sevoflurane to n-butanol. Total cholesterol(TC) is represented here as a solid line. Phospholipid (PPL) isrepresented with the solid line with solid circles. Apo B is representedby the line with solid squares. Apo A-1 is represented as a solid linewith closed triangle symbols. Apo A-2 is shown as a dashed line with astar symbol in a solid square.

FIG. 14 is a schematic representation of a Superose FPLC profile of theeffect of treatment of an aliquot of control plasma sample (FIG. 11)with a solvent ratio of 75:25 DIPE to n-butanol. Total cholesterol (TC)is represented here as a solid line. Phospholipid (PPL) is representedwith the solid line with solid circles. Apo B is represented by the linewith solid squares. Apo A-1 is represented as a solid line with closedtriangle symbols. Apo A-2 is shown as a dashed line with a star symbolin a solid square.

FIG. 15 is a schematic representation of a Superose FPLC profile of theeffect of treatment of an aliquot of control plasma sample (FIG. 11)with a solvent ratio of 95:5 DIPE to n-butanol. Total cholesterol (TC)is represented here as a solid line. Phospholipid (PPL) is representedwith the solid line with solid circles. Apo B is represented by the linewith solid squares. Apo A-1 is represented as a solid line with closedtriangle symbols. Apo A-2 is shown as a dashed line with a star symbolin a solid square.

FIG. 16 demonstrates the effects of various solvent treatments of plasmaon the ability of treated plasma to stimulate cholesterol efflux inmetabolic pathway ABCA1 and metabolic pathway SRB1 as represented incell lines COS+ and FU5AH when compared to untreated or sham treatedsamples.

FIG. 17 is a representation of Apo A-1-containing HDL subspeciesdetermined by 3-16% native PAGE, immunoblot and image analysis of asample of normal lipemic plasma (left panel) and an aliquot of thisplasma treated with sevoflurane:n-butanol (95:5) (right panel). The leftpanel depicts a distribution of protein of various HDL species having adistribution primarily comprising alpha HDL and the right panel depictsa distribution of protein of modified HDL having a distributionprimarily comprising pre-β HDL.

FIG. 18 is a representation of Apo A-1-containing HDL subspeciesdetermined by 3-16% native PAGE, immunoblot and image analysis of asample of normal lipemic plasma (left panel) and a aliquot of thisplasma treated with DIPE:n-butanol (95:5) (right panel). The left paneldepicts a distribution of protein of various HDL species having adistribution primarily comprising alpha HDL and the right panel depictsa distribution of protein of modified HDL having a distributionprimarily comprising pre-β HDL.

FIG. 19 is a schematic representation of a plurality of components usedin the present invention to achieve the novel delipidation processesdisclosed herein.

FIG. 20 is one embodiment of a configuration of a plurality ofcomponents used in the present invention to achieve the noveldelipidation processes disclosed herein.

FIG. 21 is another embodiment of a configuration of a plurality ofcomponents used in the present invention to achieve the noveldelipidation processes disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to systems, apparatus and methods useful forremoving lipid from HDL particles derived primarily from plasma ofpatients thereby creating modified HDL particles with reduced lipidcontent, particularly reduced cholesterol content. The present methodscreate these modified HDL particles with reduced lipid content withoutsubstantially modifying LDL particles.

The present invention further provides a biological fluid comprising amodified protein distribution wherein the biological fluid had a firststate, the first state having alpha high density lipoproteins andpre-beta high density lipoproteins, and wherein the biological fluid hasa second state, the second state having an increased concentration ofpre-beta high density lipoprotein relative to the first state, afterbeing exposed to a lipid removing agent. The present invention providesa biological fluid capable of enhancing an ABCA1 pathway of a patientwherein the biological fluid is made by modifying a fluid having a firstconcentration of pre-beta high density lipoproteins relative to totalprotein, wherein the modification increases the concentration ofpre-beta high density lipoprotein relative to total protein.

The present invention provides newly formed derivatives of HDL particlesthat may be administered to patients to enhance cellular cholesterolefflux and treat diseases, particularly arteriosclerosis,atherosclerosis, cardiovascular and other lipid-associated diseases.

Definitions

The term “fluid” is defined as fluids from animals or humans thatcontain lipids or lipid containing particles, fluids from culturingtissues and cells that contain lipids and fluids mixed withlipid-containing cells. For purposes of this invention, decreasing theamount of lipids in fluids includes decreasing lipids in plasma andparticles contained in plasma, including but not limited to HDLparticles. Fluids include, but are not limited to: biological fluids;such as blood, plasma, serum, lymphatic fluid, cerebrospinal fluid,peritoneal fluid, pleural fluid, pericardial fluid, various fluids ofthe reproductive system including, but not limited to, semen,ejaculatory fluids, follicular fluid and amniotic fluid; cell culturereagents such as normal sera, fetal calf serum or serum derived from anyanimal or human; and immunological reagents, such as variouspreparations of antibodies and cytokines from culturing tissues andcells, fluids mixed with lipid-containing cells, and fluids containinglipid-containing organisms, such as a saline solution containinglipid-containing organisms. A preferred fluid treated with the methodsof the present invention is plasma.

The term “lipid” is defined as any one or more of a group of fats orfat-like substances occurring in humans or animals. The fats or fat-likesubstances are characterized by their insolubility in water andsolubility in organic solvents. The term “lipid” is known to those ofordinary skill in the art and includes, but is not limited to, complexlipid, simple lipid, triglycerides, fatty acids, glycerophospholipids(phospholipids), true fats such as esters of fatty acids, glycerol,cerebrosides, waxes, and sterols such as cholesterol and ergosterol.

The term “extraction solvent” is defined as one or more solvents usedfor extracting lipids from a fluid or from particles within the fluid.This solvent enters the fluid and remains in the fluid until removed byother subsystems. Suitable extraction solvents include solvents thatextract or dissolve lipid, including but not limited to phenols,hydrocarbons, amines, ethers, esters, alcohols, halohydrocarbons,halocarbons, and combinations thereof. Preferred extraction solvents areethers, esters, alcohols, halohydrocarbons, or halocarbons whichinclude, but are not limited to di-isopropyl ether (DIPE), which is alsoreferred to as isopropyl ether, diethyl ether (DEE), which is alsoreferred to as ethyl ether, lower order alcohols such as butanol,especially n-butanol, ethyl acetate, dichloromethane, chloroform,isofluorane, sevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane-d3), perfluorocyclohexanes, trifluoroethane, cyclofluorohexanol,and combinations thereof.

The term “patient” refers to animals and humans, which may be either afluid source to be treated with the methods of the present invention ora recipient of derivatives of HDL particles and or plasma with reducedlipid content.

The term “HDL particles” encompasses several types of particles definedbased on a variety of methods such as those that measure charge,density, size and immunoaffinity, including but not limited toelectrophoretic mobility, ultracentrifugation, immunoreactivity andother methods known to one of ordinary skill in the art. Such HDLparticles include but are not limited to the following: VLDL, α HDL,pre-β HDL (including pre-β₁ HDL, pre-β₂ HDL and pre-β₃HDL), β HDL, HDL₂(including HDL_(2a) and HDL_(2b)) HDL₃, VHDL, LpA-I, LpA-II,LpA-I/LpA-II (for a review see Barrans et al., Biochemica BiophysicaActa 1300; 73-85, 1996). Accordingly, practice of the methods of thepresent invention creates modified HDL particles. These modifiedderivatives of HDL particles may be modified in numerous ways includingbut not limited to changes in one or more of the following metabolic andor physico-chemical properties (for a review see Barrans et al.,Biochemica Biophysica Acta 1300; 73-85, 1996): molecular mass (kDa);charge; diameter; shape; density; hydration density; flotationcharacteristics; content of cholesterol; content of free cholesterol;content of esterified cholesterol; molar ratio of free cholesterol tophospholipids; immunoaffinity; content, activity or helicity of one ormore of the following enzymes or proteins (Apo A-1, Apo A-2, Apo D, ApoE, Apo J, Apo A-IV, cholesterol ester transfer protein (CETP),lecithin:cholesterol acyltransferase (LCAT); capacity and/or rate forcholesterol binding, capacity and/or rate for cholesterol transport.

Methods

The methods of the present invention employ techniques to create HDLparticles with reduced lipid content. These HDL particles are obtainedfrom fluids, such as plasma. The first method comprises removal of LDLfrom plasma before treating the plasma to decrease lipids and to createHDL particles with reduced lipid content. The second method does notremove LDL from plasma before exposure to solvents but employs varioussolvent systems for enabling selective removal of lipids from HDLparticles without substantially affecting LDL. The various stepsinvolved in the two methods are described generally below. Followingthese general descriptions are descriptions of various embodiments ofthe methods of the present invention, including variants such assolvents employed, mixing methods, mixing times, and optionally,temperature.

The present invention further provides a method of modifying a proteindistribution in a fluid wherein the protein distribution has a firststate, said first state having alpha high density lipoproteins andpre-beta high density lipoproteins, comprising the steps of: exposingsaid fluid to a lipid removing agent wherein the exposure modifies theprotein distribution from the first state into a second state, saidsecond state having an increased concentration of pre-beta high densitylipoprotein relative to said first state; and removing said lipidremoving agent from the biological fluid.

The present invention also provides a method of enhancing an ABCA1pathway of a patient with a first protein distribution, the firstprotein distribution having a concentration of pre-beta high densitylipoproteins relative to total protein, comprising the step of modifyinga fluid containing the first protein distribution by exposing the fluidto a lipid removing agent, wherein the modification increases theconcentration of pre-beta high density lipoprotein relative to totalprotein, and introducing the fluid into the patient.

As discussed above, the methods and systems of the present invention maybe composed of numerous configurations. Set forth below are numerouscomponents that may be combined to create the numerous embodiments thatare capable of achieving the objectives and advantages described above.These embodiments are described to teach the invention and are not meantto limit the scope of the invention. Rather, each embodiment is but oneof many possible configurations that can be used to accomplish theobjectives described above.

LDL Extraction and Removal of Lipids from HDL Particles

In one embodiment of the present invention, as shown in FIG. 1, the HDLand LDL particles are separated prior to treatment. FIG. 1 is a flowchart of the process for LDL extraction and removal of lipids from HDLparticles.

In step 100 of the process for LDL extraction and removal of lipids fromHDL particles, the plasma is separated from the blood. In a preferredembodiment, this is achieved via filtration. In another preferredembodiment, the plasma and blood components are separated viacentrifugation. The blood can optionally be combined with ananticoagulant, such as sodium citrate, and centrifuged at forcesapproximately equal to 2,000 times gravity. The red blood cells are thenaspirated from the plasma. In step 102, the cells are returned to thepatient. In this particular embodiment of the present invention, the LDLis separated from the plasma in step 104. This is achieved via use of anaffinity column, ultracentrifugation, or any other method known to oneof ordinary skill in the art. An exemplary method is the use ofultracentrifugation, in which the plasma is passed through theultracentrifugal separator, thereby parsing out the LDL and HDLparticles. The ultracentrifugal separator uses density gradientultracentrifugation—a sophisticated and highly accurate process thatseparates lighter portions of lipoprotein from heavier portions bycentrifugal force. The LDL is discarded in step 106.

In step 108, solvents are added to the plasma which still contains HDLin order to remove lipids. The solvent types, ratios, and concentrationscan vary. The plasma and solvent are introduced into at least oneapparatus for mixing, agitating, or otherwise contacting the plasma withthe solvent. The plasma may be transported using a continuous or batchprocess. Furthermore various sensing means may be included to monitorpressures, temperatures, flow rates, solvent levels, and the like(discussed in more detail below).

In step 110, energy is introduced to the system. The various forms ofenergy employed involve mixing methods, time, and speed (variants ofwhich are discussed in further detail below). Centrifugation is employedin step 112 to remove the residual bulk solvent. The remaining solublesolvent is removed in step 114. This is achieved via charcoaladsorption, evaporation, or HFC pervaporation, as discussed below. Inoptional step 116, the mixture is tested for residual solvent via gaschromatography (GC) or any other similar means. Optionally, this step iseliminated with statistical validation. In step 118, the plasma withreduced lipid content is returned to the patient. This plasma with atleast partially or substantially reduced lipid levels, which wasseparated initially, is then treated appropriately and subsequentlyreintroduced into the body.

Selective Removal of Lipid from HDL and Formation of Modified HDLParticles

FIG. 2 presents a flowchart delineating the steps of the anotherpreferred embodiment of the present invention. In step 200, plasma isseparated from the blood via filtration, centrifugation or any othermeans known to one of ordinary skill in the art. In a preferredembodiment, the blood is passed through a centrifugal separator, whichseparates the blood into blood cells and plasma. In step 202, the cellsare returned to the patient. Solvents are added to the separated plasmain step 204 in order to extract lipids. The solvent system is optimallydesigned such that only the HDL particles are treated to reduce theirlipid levels and LDL remains at least substantially intact. The solventsystem includes factoring in variables such as solvent employed, mixingmethod, time, and temperature. Solvent type, ratios and concentrationsmay vary in this step. The plasma and solvent are introduced into atleast one apparatus for mixing, agitating, or otherwise contacting theplasma with the solvent. The plasma may be transported using acontinuous or batch process. Further, various sensing means may beincluded to monitor pressures, temperatures, flow rates, solvent levels,and the like (discussed in more detail below).

In step 206, energy is introduced into the system in the form of variedmixing methods, time, and speed. Bulk solvents are removed in step 208via centrifugation. In step 210 the remaining soluble solvent is removedvia charcoal adsorption, evaporation, or HFC pervaporation. In step 212,the mixture is optionally tested for residual solvent via use of GC, orsimilar means. The test for residual solvent may optionally beeliminated based on statistical validation. In step 214, the treatedplasma (preferably containing modified HDL particles with reduced lipidcontent), which was separated initially, is treated appropriately andsubsequently returned to the patient.

One of ordinary skill in the art would appreciate that although theprocesses shown in FIGS. 1 and 2 depict only the main steps of theprocesses and reference primary elements of the systems, they mayoptionally contain other elements such as a blood pump for maintainingproper blood volume, a blood pressure meter, a blood anticoagulant agentinjecting device, a drip chamber for eliminating air bubbles in theblood, and a heater or cooler for maintaining an appropriate temperaturefor the blood while it is outside the body.

Variables to be Considered in Employing the Methods of the PresentInvention

The present invention employs one of many optimally configured solventsystems designed to remove lipids from HDL particles while notsubstantially affecting LDL. In the first embodiment, LDL is removedfrom the plasma before treating the plasma with solvent(s) to create HDLparticles with reduced lipid content while retaining the composition ofthe plasma proteins. In the second embodiment, care is taken toselectively remove lipids from HDL particles without substantiallyaffecting LDL particles. These variables include solvent choice, mixingmethods, time, and temperature.

Plasma Separation Procedures

Typical plasma separation procedures are well known to those of ordinaryskill in the art and preferably include, but are not limited to,filtration, centrifugation, and aspiration.

LDL Extraction

Methods of LDL extraction are well known to those of ordinary skill inthe art. For purposes of the present invention, two preferred methodsare, but not limited to, use of an affinity column andultracentrifugation. The ultracentrifugal separator uses densitygradient ultra centrifugation—a sophisticated and highly accurateprocess that separates lighter portions of lipoprotein from heavierportions by centrifugal force.

Solvents Employed in the Process of Removing Lipids

Numerous organic solvents may be used in the method of this inventionfor removal of lipid from fluids and HDL particles, provided that thesolvents are effective in solubilizing lipids. Suitable solventscomprise mixtures of aromatic, aliphatic, or alicyclic hydrocarbons,ethers, phenols, esters, alcohols, halohydrocarbons, and halocarbons.Preferred solvents are ethers, for example di-isopropyl ether (DIPE).Asymmetrical ethers and halogenated ethers may be used. Particularlypreferred, as at least one component, are the C₄-C₈ containing-ethers,including but not limited to, diethyl ether, and propyl ethers,including but not limited to DIPE. Also useful in the present inventionare combinations of ethers, such as DIPE and diethyl ether. Also usefulin the present invention are combinations of ethers and alcohols, suchas DIPE and butanol. Also preferred in the present invention arecombinations of fluoroethers and alcohols, such as sevoflurane andbutanol, particularly sevoflurane and n-butanol.

Hydrocarbons in their liquid form dissolve compounds of low polaritysuch as the lipids in fluids. Accordingly, hydrocarbons comprise anysubstantially water immiscible hydrocarbon, which is liquid at about 37°C. Suitable hydrocarbons include, but are not limited to the following:C₅ to C₂₀ aliphatic hydrocarbons such as petroleum ether, hexane,heptane, and octane; haloaliphatic hydrocarbons such as chloroform,1,1,2-trichloro-1,2,2-trifluoroethane, 1,1,1-trichloroethane,trichloroethylene, tetrachloroethylene dichloromethane and carbontetrachloride; thioaliphatic hydrocarbons; perfluorocarbons, such asperfluorocyclohexane, perfluoromethylcyclohexane, andperfluorodimethylcyclohexane; fluoroethers such as sevoflurane; each ofwhich may be linear, branched or cyclic, saturated or unsaturated;aromatic hydrocarbons such as benzene; alkylarenes such as toluene,haloarenes, haloalkylarenes and thioarenes. Other suitable solvents mayalso include: saturated or unsaturated heterocyclic compounds such aswater insoluble derivatives of pyridine and aliphatic, thio or haloderivatives thereof; and perfluorooctyl bromide. Another suitablesolvent is perfluorodecalin.

Suitable esters which may be used include, but are not limited to, ethylacetate, propylacetate, butylacetate and ethylpropionate. Suitableexemplary ketones which may be used include, but are not limited to,methyl ethyl ketone.

Suitable surfactants which may be used, include but are not limited tothe following: sulfates, sulfonates, phosphates (includingphospholipids), carboxylates, and sulfosuccinates. Some anionicamphiphilic materials useful with the present invention include but arenot limited to the following: sodium dodecyl sulfate (SDS), sodium decylsulfate, bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), cholesterolsulfate and sodium laurate.

The alcohols which are preferred for use in the present invention, whenused alone, include those alcohols which are not appreciably misciblewith plasma or other biological fluids. When alcohols are used incombination with another solvent, for example, ether, a hydrocarbon, anamine or a combination thereof, C₁-C₈ containing alcohols may be used.Preferred alcohols for use in combination with another solvent includelower alcohols such as C₄-C₈ containing alcohols. Accordingly, preferredalcohols that fall within the scope of the present invention arepreferably butanols, pentanols, hexanols, heptanols and octanols, andiso forms thereof. Particularly preferred are the butanols (1-butanoland 2-butanol), also referred to as n-butanol. As stated above, the mostpreferred alcohol is the C₄ alcohol, butanol. The specific choice ofalcohol will depend on the second solvent employed. In a preferredembodiment, lower alcohols are combined with lower ethers.

Ethers, used alone, or in combination with other solvents, preferablyalcohols, are another preferred solvent for use in the method of thepresent invention. Particularly preferred are the C₄-C₈ ethers,including but not limited to, ethyl ether, diethyl ether, and propylethers, including but not limited to diisopropyl ether (DIPE). Alsouseful in the present invention are combinations of ethers, such asdi-isopropyl ether and diethyl ether. When ethers and alcohols are usedin combination as a first solvent for removing lipid, any combination ofalcohol and ether may be used provided the combination is effective topartially or completely remove lipid. When alcohols and ether arecombined as a solvent for removing lipid from a fluid, acceptable ratiosof alcohol to ether in this solvent are about 0.01 parts to 99.99 partsalcohol to about 99.99 parts to 0.01 parts ether, including a ratio ofabout 1 part to 25 parts alcohol with about 75 parts to 99 parts ether,a ratio of about 3 parts to 10 parts alcohol with about 90 parts to 97parts ether, and a preferred ratio of 5 parts alcohol with 95 partsether. An especially preferred combination of alcohol and ether is thecombination of butanol and di-isopropyl ether.

In sum, the particularly preferred solvents include 100 partsdi-isopropyl ether, a combination of 95 parts di-isopropyl ether per 5parts n-butanol, and a combination of 95 parts sevoflurane per 5 partsn-butanol. Acceptable ranges of sevoflurane and n-butanol also includeabout 0.01 parts to 99.99 parts sevoflurane per about 99.99 parts to0.01 parts n-butanol, 0.1 parts to 99.9 parts sevoflurane per about 99.9parts to 0.1 parts n-butanol; 1.0 parts to 99.0 parts sevoflurane perabout 99.0 parts to 1.0 parts n-butanol, 10.0 parts to 90.0 partssevoflurane per about 90.0 parts to 10.0 parts n-butanol, 15.0 parts to85.0 parts sevoflurane per about 85.0 parts to 15.0 parts n-butanol.Preferred combinations include about 95 parts sevoflurane per about 5.0parts n-butanol, about 90 parts sevoflurane per about 10 partsn-butanol, about 85 parts sevoflurane per about 15 parts n-butanol, and,more particularly, 97.5 parts sevoflurane per 2.5 parts n-butanol.

Acceptable ratios of solvent to plasma include any combination ofsolvent and plasma. Most preferred ratios are 2 parts plasma to 1 partsolvent, 1 part plasma to 1 part solvent, and 1 part plasma to 2 partssolvent. For example, when using a solvent comprising 95 partssevoflurane to 5 parts n-butanol, it is preferred to use two partssolvent per one part plasma.

Additionally, when employing a solvent containing n-butanol, the presentinvention can also use a ratio of solvent to plasma that yields at least3% n-butanol in the final solvent/plasma mixture. A particularlypreferred final concentration of n-butanol in the final solvent/plasmamixture is 3.33%.

Processes to Remove Lipids from Fluids and HDL Particles

The processes employed in the methods of the present invention to reducelipids in fluids and HDL particles relate directly to energy input. Theprocedure employed must be designed such that HDL particles are treatedto reduce their lipid levels without destruction of plasma proteins orsubstantially affecting LDL particles. Note that the methods describedbelow may be used to achieve the steps of both of the preferredembodiments of the present invention as described above.

Mixing Methods

The plasma and the solvent are subjected to at least one mixing methodfor mixing, agitating or otherwise contacting the biological fluid withthe solvent. The mixing method employed in the present invention may beone of, but is not limited to, an in-line static mixer, a rotatingflask, a vortexer, a centrifuge, a sonicated flask, a high shear tube, ahomogenizer, a blender, hollow fiber contactor, a centrifugal pump, ashaker table, a swirling process, a stirring process, an end-over-endrotation of a sealed container, or other suitable devices, or anycombination of these devices or processes.

Mixing Duration

The amount of time required for adequate mixing of the solvent with thefluid is related to the mixing method employed. Fluids are mixed for aperiod of time sufficient to permit intimate contact between the organicand aqueous phases, and for the solvent to at least partially orcompletely solubilize the lipid. Another consideration is temperature.The balance between the mixing time and temperature must be designedsuch that it does not encourage the contamination of or deterioration ofthe blood sample. The time and temperature system is ideally balancedsuch that the blood sample is still viable and does not deteriorate.

Typically, mixing will occur for a period of about 1 second to about 24hours, possibly about 1 second to about 2 hours, possibly approximately1 second to approximately 10 minutes, or possibly about 30 seconds toabout 1 hour, depending on the mixing method employed. Non-limitingexamples of mixing durations associated with different methodsinclude 1) gentle stirring and end-over-end rotation for a period ofabout 1 second to about 24 hours, 2) vigorous stirring and vortexing fora period of about 1 second to about 30 minutes, 3) swirling for a periodof about 1 second to about 2 hours, or 4) homogenization for a period ofabout 1 second to about 10 minutes.

Temperature

As described above, temperature is also an important consideration. Thetemperature is usually set at less that 37° C. so as not to denature theplasma. Optionally, cooler temperatures may also be employed. There arevarious methods for achieving temperature regulation in this system.

Solvent Extraction Methods

Removal of Residual Bulk Solvent

In a preferred embodiment of the present invention, the residual bulksolvent is removed via centrifugation.

Removal of Remaining Soluble Solvent

Another preferred method of separating solvent is through the use ofcharcoal, preferably activated charcoal. This charcoal is optionallycontained in a column. Alternatively, the charcoal may be in slurryform. Various biocompatible forms of charcoal may be used in thesecolumns.

HFC Pervaporation

Hollow fiber contactors (HFCs) can successfully reduce totalconcentrations of solvents, such as di-isopropyl ether and di-ethylether, in water and plasma, using different HFCs, pressures, and flowrates. HFCs may have a total surface area of permeable membrane formedby the hollow fibers between about 4,200 square centimeters and about18,000 square centimeters, depending on the type of HFC used. Further,the gas flow rate was varied in these experiments from between about 2liters per minute to about 10 liters per minute, and the plasma flowrate was varied from between about 10 mL per minute to about 60 mL perminute. Operation in this manner can reduce the initial concentrationsof solvents from between about 28,000 parts per million (ppm) and 9,000ppm to between about 1327 ppm and about 0.99 ppm within between about 14minutes and 30 minutes.

In one embodiment of the solvent removal system of the presentinvention, the solvent-treated plasma containing residual solublesolvent is typically first introduced into a circulation loop including,for instance, a recirculating vessel, a fluid transport means such astubing, valves, a pump, and a solvent extracting device, such as a HFC.In this circulation loop, the HFC functions as a recirculating,solvent-extraction device. The plasma/solvent is circulated through thehollow fiber of the HFC, thereby contacting the extraction solvent witha gas or a second extraction solvent, circulating through the shell ofthe HFC. If a volatile solvent is used as the first extraction solvent,any gas capable of extracting the first extraction solvent from thedelipidated plasma may be used, including, but not limited to, nitrogenand air.

Specific Embodiments of the Present Invention

The above-described components can be integrated into a plurality ofdifferent embodiments to enable the practice of the present invention.Certain specific embodiments shall be described herein to particularlyhighlight defined approaches to practicing the present invention. Theembodiments listed below do not represent every variation of the presentinvention and are designed to exemplify the present invention and, incertain cases, represent preferred approaches to practicing the presentinvention.

Referring to FIGS. 19 through 21, a plurality of embodiments depictingdifferent systems capable of practicing the present invention are shown.It should be understood that each embodiment has different advantagesand disadvantages, from a cost and usage perspective, and that none ofthe embodiments are specifically preferred relative to otherembodiments. FIG. 19 depicts a basic component flow diagram definingelements of the HDL modification system 1900. A fluid input is provided1905 and connected via tubing to a mixing device 1920. A solvent inputis provided 1910 and also connected via tubing to a mixing device 1920.Preferably valves 1915 are used to control the flow of fluid from fluidinput 1905 and solvent from solvent input 1910. It should be appreciatedthat the fluid input 1905 preferably contains any fluid that includesHDL particles, including plasma having LDL particles or devoid of LDLparticles, as discussed above. It should further be appreciated thatsolvent input 1910 can include a single solvent, a mixture of solvents,or a plurality of different solvents that are mixed at the point ofsolvent input 1910. While depicted as a single solvent container,solvent input 1910 can comprise a plurality of separate solventcontainers. The types of solvents that are used and preferred arediscussed above.

The mixer 1920 mixes fluid from fluid input 1905 and solvent fromsolvent input 1910 to yield a fluid-solvent mixture. Preferably, mixer1920 is capable of using a shaker bag mixing method with the input fluidand input solvent in a plurality of batches, such as 1, 2, 3 or morebatches. An exemplary mixer is a Bamstead Labline orbital shaker table.Once formed, the fluid-solvent mixture is directed, through tubing andcontrolled by at least one valve, to a separator 1925. In a preferredembodiment, separator 1925 is capable of performing bulk solventseparation through gravity separation in a funnel-shaped bag.

In the separator 1925, the fluid-solvent mixture separates into a firstlayer and second layer. The first layer comprises a mixture of solventand lipid that has been removed from the HDL particles. The second layercomprises a mixture of residual solvent, modified HDL particles, andother elements of the input fluid. One of ordinary skill in the artwould appreciate that the composition of the first layer and the secondlayer would differ based upon the nature of the input fluid. Once thefirst and second layers separate in separator 1925, the second layer istransported through tubing to a solvent extraction device 1940.Preferably, a pressure sensor 1930 and valve is positioned in the flowstream to control the flow of the second layer to the solvent extractiondevice 1940.

The opening and closing of valves to enable the flow of fluid from inputcontainers 1905, 1910 is preferably timed using mass balancecalculations derived from weight determinations of the fluid inputs1905, 1910 and separator 1925. For example, the valves between separator1925 and waste container 1935 and between separator 1925 and solventextraction device 1940 open after the input masses (fluid and solvent)substantially balances with the mass in separator 1925 and a sufficientperiod of time has elapsed to permit separation between the first andsecond layers, as discussed above. Depending on what solvent is used,and therefore which layer settles to the bottom of the separator 1925,either the valve between separator 1925 and waste container 1935 isopened or between separators 1925 and solvent extraction device 1940 isopened. One of ordinary skill in the art would appreciate that thetiming of the opening is dependent upon how much fluid is in the firstand second layers and would further appreciate that it is preferred tokeep the valve between separator 1925 and waste container 1935 open justlong enough to remove all of the first layer and some of the secondlayer, thereby ensuring that as much solvent as possible has beenremoved from the fluid being sent to the solvent extraction device 1940.

Preferably, a glucose input 1955 and saline input 1960 is in fluidcommunication with the fluid path leading from the separator 1925 to thesolvent extraction device 1940. A plurality of valves is also preferablyincorporated in the flow stream from the glucose input 1955 and salineinput 1960 to the tubing providing the flow path from the separator 1925to the solvent extraction device 1940. Glucose and saline areincorporated into the present invention in order to prime the solventextraction device 1940 prior to operation of the system. Where suchpriming is not required, the glucose and saline inputs are not required.Also, one of ordinary skill in the art would appreciate that the glucoseand saline inputs can be replaced with other primers if the solventextraction device 1940 requires it.

The solvent extraction device 1940 is preferably a charcoal columndesigned to remove the specific solvent used in the solvent input 1910.An exemplary solvent extraction device 1940 is an Asahi Hemosorbercharcoal column. A pump 1950 is used to move the second layer from theseparator 1925, through the solvent extraction device 1940, and to anoutput container 1945. The pump is preferably a peristaltic pump, suchas a Masterflex Model 77201-62.

The first layer is directed to a waste container 1935 that is in fluidcommunication with separator 1925 through tubing and at least one valve.Additionally, other waste, if generated, can be directed from the fluidpath connecting solvent extraction device 1940 and output container 1945to waste container 1935.

Preferably, an embodiment of the present invention uses gravity,wherever practical, to move fluid through each of the plurality ofcomponents. For example, preferably gravity is used to drain the inputplasma 1905 and input solvent 1910 into the mixer 1920. Where the mixer1920 comprises a shaker bag and separator 1925 comprises a funnel bag,fluid is moved from the shaker bag to the funnel bag and, subsequently,to the waste container 1935, if appropriate, using gravity.

In an additional step, not shown in FIG. 19, the output fluid in outputcontainer 1945 would be subjected to a solvent detection system, orlipid removing agent detection system, to determine if any solvent, orother undesirable component, is in the output fluid. In one embodiment,the output fluid is subjected to sensors that are capable of determiningthe concentrations of solvents introduced in the solvent input, such asn-butanol or di-isopropyl ether. This is an important measurementbecause the output fluid is returned to the bloodstream of the patientand the solvent concentrations must be below a predetermined level tocarry out this operation safely. The sensors are preferably capable ofproviding such concentration information on a real-time basis andwithout having to physically transport a sample of the output fluid, orair in the headspace, to a remote device.

In one embodiment, molecularly imprinted polymer technology is used toenable surface acoustic wave sensors. A surface acoustic wave sensorreceives an input, through some interaction of its surface with thesurrounding environment, and yields an electrical response, generated bythe piezoelectric properties of the sensor substrate. To enable theinteraction, molecularly imprinted polymer technology is used.Molecularly imprinted polymers are plastics programmed to recognizetarget molecules, like pharmaceuticals, toxins or environmentalpollutants, in complex biological samples. The molecular imprintingtechnology is enabled by the polymerization of one or more functionalmonomers with an excess of a crosslinking monomer in presence of atarget template molecule exhibiting a structure similar to the targetmolecule that is to be recognized, i.e. the target solvent.

The use of molecularly imprinted polymer technology to enable surfaceacoustic wave sensors is preferred relative to other technologicalapproaches because they can be made more specific to the concentrationsof targeted solvents and are capable of differentiating such targetedsolvents from other possible interferents. As a result, the presence ofacceptable interferents that may have similar structures and/orproperties to the targeted solvents would not prevent the sensor fromaccurately reporting existing respective solvent concentrations.

Alternatively, if the input solvent comprises certain solvents, such asn-butanol, electrochemical oxidation could be used to measure thesolvent concentration. Electrochemical measurements have severaladvantages. They are simple, sensitive, fast, and have a wide dynamicrange. The instrumentation is simple and not affected by humidity. Inone embodiment, the target solvent, such as n-butanol, is oxidized on aplatinum electrode using cyclic voltammetry. This technique is based onvarying the applied potential at a working electrode in both the forwardand reverse directions, at a predefined scan rate, while monitoring thecurrent. One full cycle, a partial cycle, or a series of cycles can beperformed. While platinum is the preferred electrode material, otherelectrodes, such as gold, silver, iridium, or graphite, could be used.Although, cyclic voltammetric techniques are used, other pulsetechniques such as differential pulse voltammetry or square wavevoltammetry may increase the speed and sensitivity of measurements. Thealternative, a Taguchi sensor, is not preferred because it does noteffectively operate in humid conditions.

The present invention expressly covers any and all forms ofautomatically sampling and measuring, detecting, and analyzing an outputfluid, or the headspace above the output fluid. For example, suchautomated detection can be achieved by integrating a mini-gaschromatography (GC) measuring device that automatically samples air inthe output container, transmits it to a GC device optimized for thespecific solvents used in the delipidation process, and, using known GCtechniques, analyzes the sample for the presence of the solvents.

Referring back to FIG. 19, suitable materials for use in any of theapparatus components as described herein include materials that arebiocompatible, approved for medical applications that involve contactwith internal body fluids, and in compliance with U.S. PV1 or ISO 10993standards. Further, the materials should not substantially degrade from,for instance, exposure to the solvents used in the present invention,during at least a single use. The materials should typically besterilizable either by radiation or ethylene oxide (EtO) sterilization.Such suitable materials should be capable of being formed into objectsusing conventional processes, such as, but not limited to, extrusion,injection molding and others. Materials meeting these requirementsinclude, but are not limited to, nylon, polypropylene, polycarbonate,acrylic, polysulphone, polyvinylidene fluoride (PVDF), fluoroelastomerssuch as VITON, available from DuPont Dow Elastomers L.L.C.,thermoplastic elastomers such as SANTOPRENE, available from Monsanto,polyurethane, polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE),polyphenylene ether (PFE), perfluoroalkoxy copolymer (PFA), which isavailable as TEFLON PFA from E.I. du Pont de Nemours and Company, andcombinations thereof.

The valves used in each embodiment may be composed of, but are notlimited to, pinch, globe, ball, gate or other conventional valves.Preferably, the valves are occlusion valves such as Acro Associates'Model 955 valve. However, the invention is not limited to a valve havinga particular style. Further, the components of each system describedbelow may be physically coupled together or coupled together usingconduits that may be composed of flexible or rigid pipe, tubing or othersuch devices known to those of ordinary skill in the art.

Referring to FIG. 20, a specific configuration 2000 of the presentinvention is shown. A preferred configuration 2000 comprises an enclosedhousing 2005 capable of safely containing volatile fluids, such assolvents. In a preferred embodiment, the enclosed housing 2005 comprisesa door 2015 with a clear door to observe the delipidation process inoperation, a mobile base 2025, a control display 2020, a clear top 2010,and a filter and air circulating system [not shown]. The interfacescreen of the control display 2020 is preferably functional when thedoor 2015 is open to enable system set up and priming a solventextraction device but does not permit the system to delipidate an inputfluid until the door 2015 is closed and, preferably, locked. It is alsopreferred to have a waste or overflow tray capable of trapping any fluidleaks, overflows, or other spills at the base of the enclosed housing2005 in a manner that permits the tray to be readily removed withoutopening the housing 2005.

Referring to FIG. 21, a configuration of basic components of the HDLmodification system 2100 is shown. A fluid input is provided 2105 andconnected via tubing to a mixing device 2120. A solvent input isprovided 2110 and also connected via tubing to a mixing device 2120.Preferably valves are used to control the flow of fluid from fluid input2105 and solvent from solvent input 2110. It should be appreciated thatthe fluid input 2105 preferably contains any fluid that includes HDLparticles, including plasma having LDL particles or devoid of LDLparticles, as discussed above. It should further be appreciated thatsolvent input 2110 can include a single solvent, a mixture of solvents,or a plurality of different solvents that are mixed at the point ofsolvent input 2110. While depicted as a single solvent container,solvent input 2110 can comprise a plurality of separate solventcontainers. The types of solvents that are used and preferred arediscussed above.

The mixer 2120 mixes fluid from fluid input 2105 and solvent fromsolvent input 2110 to yield a fluid-solvent mixture. Preferably, mixer2120 is capable of using a shaker bag mixing method with the input fluidand input solvent in a plurality of batches, such as 1, 2, 3 or morebatches. Once formed, the fluid-solvent mixture is directed, throughtubing and controlled by at least one valve, to a separator 2125. In apreferred embodiment, separator 2125 is capable of performing bulksolvent separation through gravity separation in a funnel-shaped bag.

In the separator 2125, the fluid-solvent mixture separates into a firstlayer and second layer. The first layer comprises a mixture of solventand lipid that has been removed from the HDL particles. The second layercomprises a mixture of residual solvent, modified HDL particles, andother elements of the input fluid. One of ordinary skill in the artwould appreciate that the composition of the first layer and the secondlayer would differ based upon the nature of the input fluid. Once thefirst and second layers separate in separator 2125, the second layer istransported through tubing to a solvent extraction device 2140.Preferably, a pressure sensor and valve is positioned in the flow streamto control the flow of the second layer to the solvent extraction device2140.

Preferably, a glucose input 2130 and saline input 2150 is in fluidcommunication with the fluid path leading from the separator 2125 to thesolvent extraction device 2140. A plurality of valves is also preferablyincorporated in the flow stream from the glucose input 2130 and salineinput 2150 to the tubing providing the flow path from the separator 2125to the solvent extraction device 2140. Glucose and saline areincorporated into the present invention in order to prime the solventextraction device 2140 prior to operation of the system. Where suchpriming is not required, the glucose and saline inputs are not required.Also, one of ordinary skill in the art would appreciate that the glucoseand saline inputs can be replaced with other primers if the solventextraction device 2140 requires it.

The solvent extraction device 2140 is preferably a charcoal columndesigned to remove the specific solvent used in the solvent input 2110.An exemplary solvent extraction device 2140 is an Asahi Hemosorbercharcoal column. A pump 2135 is used to move the second layer from theseparator 2125, through the solvent extraction device 2140, and to anoutput container 2115. The pump is preferably a peristaltic pump, suchas a Masterflex Model 77201-62.

The first layer is directed to a waste container 2155 that is in fluidcommunication with separator 2125 through tubing and at least one valve.Additionally, other waste, if generated, can be directed from the fluidpath connecting solvent extraction device 2140 and output container 2115to waste container 2155.

Preferably, an embodiment of the present invention uses gravity,wherever practical, to move fluid through each of the plurality ofcomponents. For example, preferably gravity is used to drain the inputplasma 2105 and input solvent 2110 into the mixer 2120. Where the mixer2120 comprises a shaker bag and separator 2125 comprises a funnel bag,fluid is moved from the shaker bag to the funnel bag and, subsequently,to the waste container 2155, if appropriate, using gravity.

In general, the present invention preferably comprises configurationswherein all inputs, such as input plasma and input solvents, disposableelements, such as mixing bags, separator bags, waste bags, solventextraction devices, and solvent detection devices, and output containersare in easily accessible positions and can be readily removed andreplaced by a technician.

To enable the operation of the above described embodiments of thepresent invention, it is preferable to supply a user of such embodimentswith a packaged set of components, in kit form, comprising eachcomponent required to practice the present invention. Such a kit wouldpreferably include an input fluid container (i.e. a high densitylipoprotein source container), a lipid removing agent source container(i.e. a solvent container), disposable components of a mixer, such as abag or other container, disposable components of a separator, such as abag or other container, disposable components of a solvent extractiondevice (i.e. a charcoal column), an output container, disposablecomponents of a waste container, such as a bag or other container,solvent detection devices, and, a plurality of tubing and a plurality ofvalves for controlling the flow of input fluid (high densitylipoprotein) from the input container and lipid removing agent (solvent)from the solvent container to the mixer, for controlling the flow of themixture of lipid removing agent, lipid, and particle derivative to theseparator, for controlling the flow of lipid and lipid removing agent toa waste container, for controlling the flow of residual lipid removingagent, residual lipid, and particle derivative to the extraction device,and for controlling the flow of particle derivative to the outputcontainer.

In one embodiment, a kit comprises a plastic container having disposablecomponents of a mixer, such as a bag or other container, disposablecomponents of a separator, such as a bag or other container, disposablecomponents of a waste container, such as a bag or other container, and,a plurality of tubing and a plurality of valves for controlling the flowof input fluid (high density lipoprotein) from the input container andlipid removing agent (solvent) from the solvent container to the mixer,for controlling the flow of the mixture of lipid removing agent, lipid,and particle derivative to the separator, for controlling the flow oflipid and lipid removing agent to a waste container, for controlling theflow of residual lipid removing agent, residual lipid, and particlederivative to the extraction device, and for controlling the flow ofparticle derivative to the output container. Disposable components of asolvent extraction device (i.e. a charcoal column), the input fluid, theinput solvent, and solvent extraction devices are provided separately.

Administration Schedule

The modified HDL particles of the present invention may be administeredaccording to any schedule that is effective in promoting cellularcholesterol efflux.

In one embodiment, blood is withdrawn from a patient in a volumesufficient to produce about 1 liter of plasma. The blood is separatedinto plasma and red blood cells using methods commonly known to one ofskill in the art, such as plasmapheresis, and the red blood cells arestored in an appropriate storage solution or returned to the patientduring plasmapheresis. The red blood cells are preferably returned tothe patient during plasmapheresis. Physiological saline is alsooptionally administered to the patient to replenish volume. The 1 literof plasma is treated with any of the methods of the present invention tocreate HDL particles with reduced lipid content while not substantiallyaffecting LDL. The resulting treated plasma containing the HDL particleswith reduced lipid and substantially unaffected LDL content isoptionally combined with the patient's red blood cells, if the red cellswere not already returned during plasmapheresis, and administered to thepatient. One route of administration is through the vascular system,preferably intravenously. This treatment regimen is repeated weekly forabout 5 to 6 weeks. Enhanced cholesterol efflux is observed in thepatient after the treatment.

In another embodiment, blood is withdrawn from a patient in a volumesufficient to produce about 1 liter of plasma. The blood is separatedinto plasma and red blood cells using methods commonly known to one ofskill in the art, such as plasmapheresis, and the red blood cells arestored in an appropriate storage solution or returned to the patientduring plasmapheresis. The 1 liter of plasma is treated to remove theLDL component before further treatment of the plasma. The 1 liter ofplasma is treated with the method of the present invention to create HDLparticles with reduced lipid content. The resulting treated plasmacontaining the HDL particles with reduced lipid is optionally combinedwith the patient's red blood cells, if the red cells were not alreadyreturned during plasmapheresis, and administered to the patient. Oneroute of administration is through the vascular system, preferablyintravenously. This treatment regimen is repeated weekly for about 5 to6 weeks.

It is to be understood that other volumes of plasma may be treated withthe method of the present invention, and administered to a patient onvarious administration schedules. For a batch process, volumes of 100 mlto 3500 ml of plasma may be treated with the present method. Thefrequency of treatment may also vary from between several times per weekto once a month or less, depending on the volume to be treated and theseverity of the condition of the patient.

In another approach of the present invention, following removal of adesired volume of a patient's blood, separation of the blood into plasmaand red blood cells and treatment of the plasma to reduce lipid levels,the HDL particles with reduced lipid content are isolated from theplasma and administered to the patient in an acceptable vehicle.

In yet another embodiment, heterologous plasma may be obtained, treatedwith the method of the present invention and the treated plasmacontaining HDL particles with reduced lipid content administered to apatient who was not the source of the plasma. In a further embodiment,heterologous plasma may be obtained, treated with the method of thepresent invention and the HDL particles with reduced lipid content areseparated from the treated plasma. These HDL particles with reducedlipid content may be administered in an acceptable vehicle to a patientwho was not the source of the plasma.

In still another embodiment, following removal of a desired volume of apatient's blood, the patient is permitted to recover for 1 to 4 days interms of producing new blood and attaining endogenous plasma HDL levelssubstantially similar to plasma HDL levels before removing the blood.The removed blood is separated into plasma and red blood cells and theplasma is treated to reduce lipid levels. The HDL particles with reducedlipid content are isolated from the plasma and administered to thepatient. Alternatively, the HDL particles with reduced lipid content arenot isolated from the plasma and the treated plasma is administered tothe patient.

In another embodiment plasma is treated with the methods of the presentinvention to reduce lipid levels. Next, Apo A-1 protein is purified fromthis treated plasma using techniques such as affinity chromatography.The resulting purified, modified Apo A-1 protein is administered in anacceptable vehicle to a patient together with the modified HDL particleswith reduced lipid content. These modified HDL particles with reducedlipid content may be provided to the patient as isolated HDL particlesin an acceptable vehicle or included with the treated plasma.

Administration with Other Therapies

The modified HDL particles of the present invention may be administeredin conjunction with one or more additional therapeutic approaches. Themodified HDL particles of the present invention may be administered inconjunction with exercise and dietary restriction of fat and cholesterolintake.

The modified HDL particles of the present invention may be administeredin conjunction with administration of agents for reducing cholesterol,reducing LDL levels and enhancing HDL levels. These agents, such asHMG-CoA reductase inhibitors, or statins, may be administered in dosagesand according to administration schedules commonly known to one ofordinary skill in the art. Statins include but are not limited tocerivastatin, atorvastatin, fluvastatin, simvastatin, pravastatin andlovastatin. For example, dosages of 10 mg, 20 mg, 40 mg or 80 mg ofstatins, taken once per day, are commonly employed. Administration ofthe modified HDL particles of the present invention can eliminate theneed for statin therapy in patients or reduce the required dosage ofstatins.

In another aspect, the modified HDL particles of the present inventionare used in conjunction with administration of agents designed to reduceabsorption of fat and cholesterol. Such agents, for example ezetimibe,and the clinically appropriate dosages are known to one of ordinaryskill in the art.

In yet another aspect, the modified HDL particles of the presentinvention are used in conjunction with administration of one or moreagents such as fibric acid derivatives (gemfibrozil), nicotinic acid(niacin), and bile acid-binding resins (cholestyramine, cholestipol),and the clinically appropriate dosages are known to one of ordinaryskill in the art.

In yet another aspect, the modified HDL particles of the presentinvention are used in conjunction with administration ofanti-inflammatory drugs, such as aspirin, known to one of ordinary skillin the art. The clinically appropriate dosages of anti-inflammatorydrugs are known to one of ordinary skill in the art. Anti-inflammatorydrugs are often prescribed to patients with vascular disease since it isbelieved that inflammation is a causative factor of atherosclerosis andother vascular diseases.

The modified HDL particles of the present invention are used inconjunction with administration of agents such as statins and withagents designed to reduce absorption of fat and cholesterol. Thiscombination of three therapies is effective in enhancing cholesterolefflux from cells and permits administration of lower dosages ofstatins. The modified HDL particles of the present invention are alsoused in conjunction with any of the therapeutic approaches describedabove.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various embodiments, modifications and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the invention.

EXAMPLE 1

Separation and Characterization of Total Cholesterol, Apolipoprotein A1(Apo A-1), Apolipoprotein B (Apo B) and Phospholipids in Normal Plasma

A 25 ml pool of plasma was characterized in terms of total cholesterol,apolipoprotein A1 (Apo A-1), apolipoprotein B (Apo B) and phospholipids.An aliquot of 1 ml of the pooled plasma was loaded onto a SephacrylS-300 26/60 (FPLC) column. An elution buffer of phosphate bufferedsaline containing 1 mM EDTA was applied to the column and eluted at 2ml/min. About 96 fractions were collected, one every 43 secondsbeginning 41 minutes after application of the plasma sample. Eachfraction was characterized in terms of total cholesterol, Apo A-1, Apo Band phospholipids.

Apo B containing particles comprised of very low density lipoprotein(VLDL), intermediate density lipoprotein (IDL) and low densitylipoprotein (LDL) particles eluted in fractions 10-40. Apo A-1containing particles comprised of high density lipoprotein (HDL)particles eluted in the remaining fractions. The results are displayedin FIG. 3. An analysis of total plasma cholesterol indicated a clearseparation of the LDL particles from the HDL particles. The distributionof Apo A-1 and Apo B in the corresponding fractions confirmed theseparation of these particles.

EXAMPLE 2

Selective Creation of Apo A-1-Associated HDL Particles with ReducedCholesterol or with Reduced Cholesterol and Phospholipids Using DIPE

This selective plasma treatment method employs a ratio of 1:1DIPE:plasma. The sample was vortexed for 15 seconds and then permittedto separate by gravity. Activated charcoal was used to remove residualDIPE after the process and several different hematological parameterswere measured.

The delipidated sample was then applied to a column and treated asexplained in Example 1. This method removed about 10% of totalcholesterol, 12% of Apo B, 17% of Apo A-1 and about 11% ofphospholipids.

A comparison of the elution of the delipidated sample with the elutionof normal, non-delipidated plasma is presented in FIGS. 4 and 5. Theresults show a shift to the right of the Apo A-1-associated HDLparticles, indicating a smaller particle not associated with cholesterol(FIG. 4) and also not associated with phospholipid (FIG. 5).Accordingly, this delipidation method created HDL particles associatedwith Apo A-1 that were low or substantially devoid of cholesterol andphospholipid and therefore had new capacity to bind with cholesterol andphospholipid. Lipid was only slightly removed from LDL particles (Apo B)with this method (FIG. 6).

In summary, two types of HDL particles were observed. There was a widerange of sizes of HDL particles containing Apo A-1 and phospholipids butno cholesterol. A relatively narrow size range of HDL particlescontaining Apo A-1 but no phospholipids or cholesterol was observed.

EXAMPLE 3

Selective Creation of Apo A-1-Associated HDL Particles with ReducedCholesterol or with Reduced Cholesterol and Phospholipids Using aSevoflurane:n-butanol Mixture.

A mixture of sevoflurane and n-butanol was employed as a solvent in aconcentration of 95% sevoflurane and 5% n-butanol. The mixture was addedto plasma in a 2:1 solvent to plasma ratio. The sample was vortexed for15 seconds and then centrifuged. Activated charcoal was added to removeresidual solvent. The resulting solvent-free sample was run on an FPLCcolumn as described in Example 1. Data concerning the percentagereduction in cholesterol, phospholipid and Apo A-1 were obtained fromquantitative measurements and not from the FPLC elution profiles.

This method reduced total cholesterol by 9% and phospholipids by 9%. Adecrease of about 14% was observed in Apo A-1. FIG. 7 shows that thismethod resulted in Apo A-1 associated HDL particles of lower weight thatwere not associated with cholesterol when compared to plasma that wasnot subjected to such treatment. However, these Apo A-1 associated HDLparticles were associated with phospholipid (FIG. 8). There was littleeffect on Apo B associated LDL particles as shown in FIG. 9. In summary,this process resulted in a modified HDL particle which contained Apo A-1and phospholipids, but little or no cholesterol.

EXAMPLE 4

Analysis of Clinical Parameters in Normal Plasma and Plasma Treated withDIPE or sevoflurane:n-butanol

Alanine aminotransferase (ALT), alkaline phosphatase (AP), bilirubin-T,sodium, potassium, phosphorus, albumin, globulin and thealbumin/globulin (A/G) ratio were analyzed in normal untreated plasmaand in plasma treated with 100% DIPE or with sevoflurane:n-butanol. Theresults are presented in FIG. 10.

Treatment with DIPE did not change the parameters to any clinicallysignificant degree. Treatment with sevoflurane:n-butanol did not changethe parameters to any clinically significant degree. These treatments,therefore, do not substantially affect non-HDL plasma constituentcomponents.

EXAMPLE 5 Summary of the Efficacy of Different Solvents on Removal ofCholesterol from HDL and Effects on LDL

FIGS. 11-15 show a Superose FPLC profile of plasma treated with nothing,DIPE (100%), sevoflurane:n-butanol (95:5), sevoflurane:n-butanol (75:25)and DIPE:n-butanol (95:5), respectively, for a variety of parameters.Shown are total cholesterol, phospholipid, Apo B, Apo A-1, and Apo A2.The data indicate that cholesterol is reduced following solventtreatment in the areas associated with Apo A-1 and Apo A2 (the peak onthe right side of each figure) while the Apo B associated with LDL(middle peak) remains substantially unchanged. However, a severe solventtreatment with a solvent ratio of 75:25 DIPE:n-butanol (FIG. 14)dramatically reduced total cholesterol and phospholipids when comparedto untreated plasma (FIG. 11).

EXAMPLE 6

Cholesterol Efflux Studies of Plasma Treated with Various Solvents

All solvent conditions above were employed to test the effects oftreated plasma on cholesterol efflux in ABCA1 pathway and SRB1 pathwayas measured in COS and Fu5AH cells. The methods employed were thosedescribed by Rothblatt and colleagues (de la Llera Moya et al.,Arteriosclerosis. & Thrombosis 14:1056-1065, 1994).

The methods employed are described generally in the next paragraphs. Thetissue culture cell system was designed to quantitate the contributionof scavenger receptor BI (SR-BI) or ATP-binding cassette transporter 1(ABCA1) to the efflux of cellular cholesterol when cells are exposed toserum or isolated lipoproteins. The general approach is to measure therelease of radiolabeled cellular cholesterol to either isolatedacceptors or whole serum. The contributions of SR-BI or ABCA1 to thisefflux process are determined by comparing the release obtained fromcells lacking the specific receptor to that observed in parallel cellcultures expressing the receptor. Thus, to quantitate the contributionof ABCA1 to cellular cholesterol efflux, transformed mouse macrophagecells are grown in monolayers and prelabeled with ³H-cholesterol. Oneset of monolayers is treated with cAMP which has been shown toupregulate the ABCA1 receptor, whereas a replicate set of monolayers theare left untreated, and serve as control cells which lack ABCA1. Thesera to be tested is diluted to an appropriate concentration andincubated with both ABCA1 positive and negative monolayers. The releaseof the radiolabeled cholesterol is determined after an appropriateincubation time ranging from 1 to 12 hours. The ABCA1-contribution toefflux is determined by subtracting the efflux obtained in ABCA1negative cultures from that obtained from the ABCA1 positive cultures.

A general assay for determining the contribution of SRBI to cholesterolefflux uses the same approach as described above. Cell lines serving ascholesterol donors are treated so that they either lack SRBI or expresshigh levels of the receptor. In the general protocol presently usedCOS-7 cells are transiently transfected when it SR-BI. These cells arepre-labeled with 3H-cholesterol and then exposed to the test serum forappropriate periods of time. Following this period the medium is removedand a determination is made of the amount of radiolabeled cellularcholesterol that has been released. The efflux of cholesterol fromcontrol, SR-BI negative cells is subtracted from that observed withSR-BI expressing cells. The difference obtained by this calculationreflects the contribution of SR-BI to cholesterol efflux. An alternativecell system that can be used for determining SR-BI-mediated efflux isthe Fu5AH rat hepatoma cell. These cells expresses very high levels ofSR-BI and the efflux of radiolabeled cholesterol from Fu5AH is a veryreliable measure of the contribution of SR-BI to the efflux process.

The results are shown in FIG. 16 and demonstrate that plasma treatedwith the various solvents stimulated efflux of cholesterol 20 to 25times more efficiently than untreated or sham treated plasma, taken fromthe same pool of starting plasma. This effect was observed in ABCA1cells, which possess a metabolic pathway believed to be representativeof cholesterol egress from arterial walls, but not in COS+ or FU5AHcells which are believed to be representative of the SRB1 pathway in theliver. Further testing of sevoflurane:n-butanol of three differentplasma samples obtained from different individuals produced similarresults (FIG. 16, set of histograms). By creating a modified HDLparticle, the present invention therefore positively affects theeffectiveness of the SRB1 and ABCA1 pathways. The present invention alsoencompasses the modification of SRB1 and ABCA1 pathways by modifying therelative ratio of phospholipid to Apo A-1 in HDL particles via the abovedescribed delipidation processes.

EXAMPLE 7

Analysis of Solvent Treatment on Levels of Apo A-1 Associated Pre B-2HDL and Pre B-1 HDL Particles

The individual effects of sevoflurane:n-butanol and DIPE:n-butanol(95:5) on Apo A-1-containing HDL subspecies were examined using 3-16%native PAGE gels, followed by immunoblotting and image analysis. Thetechniques employed are described in Asztalos et al., Arteriosclerosis,Thrombosis and Vascular Biology; 15:1419-1423, 1995 and Asztalos et al.,Arteriosclerosis, Thrombosis and Vascular Biology; 17:1885-1893, 1997.

The left side of FIGS. 17 and 18 each show Apo A-1-containing HDLsubspecies, namely preβ-2, preβ-1, a and pre-α particles in normallipemic plasma, while the right panel of each figure shows normal plasmatreated with sevoflurane:n-butanol and DIPE:n-butanol (95:5)

FIG. 17 demonstrates that plasma treated with sevoflurane:n-butanolshowed an increase in the Apo A-1-containing preβ-2, preβ-1 HDLsubspecies and a decrease in the a HDL subspecies. A similar pattern wasobserved following treatment with DIPE:n-butanol (FIG. 18). Theseresults demonstrate that these solvent treatments of plasma increasedApo A-1-containing preβ-2, preβ-1 HDL subspecies, thereby enhancingtheir availability for accepting new cholesterol and facilitatingcellular cholesterol efflux.

A similar comparison of the untreated normal plasma shown in the leftpanel of FIGS. 17 and 18 and another untreated plasma sample withslightly elevated cholesterol levels produced a similar pattern (datanot shown) with most of the immunoreactive HDL species appearing in theαHDL form with relatively minor density associated with preβ-2 andpreβ-1 HDL particles. These results provide an internal methodologicalcontrol.

EXAMPLE 8

Administration of Derivatives of HDL to a Patient with ElevatedCholesterol and Coronary Artery Disease

A 51 year old male patient presents with acute coronary syndrome and isdetermined to have atherosclerosis via angiography. A unit of blood isremoved weekly from the patient. The plasma is recovered and processedwith the method of the present invention to produce derivatives of HDLthat are particles with reduced cholesterol content while the red bloodcells are returned to the patient. These HDL particles with reducedcholesterol content in the treated plasma are administered to thepatient intravascularly at weekly intervals for 5-10 weeks. Anotherangiography test after completion of the treatment shows lower amountsof atherogenic plaque in the coronary vessels compared to the firstangiography test.

EXAMPLE 9

Administration of Derivatives of HDL Together with Atorvastatin andEzetimibe to a Patient with Elevated Cholesterol and Coronary ArteryDisease

A 58 year old female presents with acute coronary syndrome and isdetermined to have atherosclerosis via angiography. A unit of blood isremoved weekly from the patient. The plasma is recovered and processedwith the method of the present invention to produce derivatives of HDLthat are particles with reduced cholesterol content while the red bloodcells are returned to the patient. These HDL particles with reducedcholesterol content in the treated plasma are administered to thepatient intravascularly at weekly intervals for 5-10 weeks. The patienthad previously received 80 mg of atorvastatin daily with 10 mg ofezetimibe. These drugs are continued daily together with the weeklyadministration of HDL particles with reduced cholesterol content.Another angiography test after completion of the treatment shows loweramounts of atherogenic plaque in the coronary vessels compared to thefirst angiography test.

EXAMPLE 10

Administration of Derivatives of HDL Together with Simvastatin andEzetimibe to an Obese Patient with Elevated Cholesterol and CoronaryArtery Disease

A 48 year old obese female patient presents with elevated levels of LDLand cholesterol, and an angiographic test result indicatingatherosclerosis in three coronary arteries. A unit of blood is removedweekly from the patient. The plasma is recovered and processed with themethod of the present invention to produce derivatives of HDL that areparticles with reduced cholesterol content while the red blood cells arereturned to the patient. These HDL particles with reduced cholesterolcontent are combined with the treated plasma and administered to thepatient intravascularly at weekly intervals for 5-10 weeks. The patienthad previously received 80 mg of simvastatin daily with 10 mg ofezetimibe. These drugs are continued daily together with the weeklyadministration of HDL particles with reduced cholesterol content. Thepatient is placed on a moderate exercise schedule.

New blood work indicates a reduction in circulating cholesterol, areduction in LDL and an increase in circulating HDL. The patient loses15 pounds during the five month period. A new angiographic procedureshows lower amounts of atherogenic plaque in the coronary vesselscompared to the first angiographic procedure.

EXAMPLE 11

Administration of Derivatives of HDL to a Diabetic Patient with ElevatedCholesterol and Coronary Artery Disease

A 44 year old diabetic female patient presents with elevated levels ofLDL and cholesterol, and an angiographic test result indicatingatherosclerosis in two coronary arteries. A unit of blood is removedweekly from the patient. The plasma is recovered and processed with themethod of the present invention to produce derivatives of HDL that areparticles with reduced cholesterol content while the red blood cells arereturned to the patient. These HDL particles with reduced cholesterolcontent are combined with the treated plasma and administered to thepatient intravascularly at weekly intervals for 5-10 weeks. The patienthad previously received daily insulin injections. These injections arecontinued daily together with the weekly administration of HDL particleswith reduced cholesterol content.

New blood work indicates a reduction in circulating cholesterol, areduction in LDL and an increase in circulating HDL. A new angiographicprocedure shows lower amounts of atherogenic plaque in the coronaryvessels compared to the first angiographic procedure.

EXAMPLE 12

Administration of Derivatives of HDL to a Patient with ElevatedCholesterol and Peripheral Vascular Disease Causing IntermittentClaudication

A 66 year old male patient presents with elevated levels of LDL andcholesterol and reports pain in the right lower extremity. Anangiographic test indicating atherosclerosis in the right popliteal andposterior tibial arteries, leading to a diagnosis of intermittentclaudication. A unit of blood is removed weekly from the patient. Theplasma is recovered and processed with the method of the presentinvention to produce derivatives of HDL that are particles with reducedcholesterol content while the red blood cells are returned to thepatient. These HDL particles with reduced cholesterol content arecombined with the treated plasma and administered to the patientintravascularly at weekly intervals for 5-10 weeks.

New blood work indicates a reduction in circulating cholesterol, areduction in LDL and an increase in circulating HDL. A new angiographicprocedure shows lower amounts of atherogenic plaque in the rightpopliteal and posterior tibial arteries compared to the firstangiographic procedure. The patient reports decreased levels of painfrom the right lower extremity.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. The terms and expressions whichhave been employed herein are used as terms of description and not oflimitation, and there is no intention, in the use of such terms andexpressions, of excluding any equivalents of the features shown anddescribed or portions thereof. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1-50. (canceled)
 51. A method of modifying a protein distribution in afluid wherein the protein distribution has a first state, the firststate having alpha high density lipoproteins and pre-beta high densitylipoproteins, comprising the steps of: exposing the fluid to a lipidremoving agent wherein the exposure modifies the protein distributionfrom the first state into a second state, the second state having anincreased concentration of pre-beta high density lipoprotein relative tothe first state; and, removing the lipid removing agent from thebiological fluid.
 52. The biological fluid of claim 51 wherein thebiological fluid is plasma. 53-72. (canceled)